The invention relates to chemical compounds for delivering therapeutic agents to tissues of a mammal, such as a human. More particularly, the invention involves a carrier for delivery of a therapeutic agent in which a biodegradable disulfide bond conjugates the therapeutic agent to a carrier of the present invention. The disulfide bond can be reduced in an appropriate environment in vivo so that a greater amount of the therapeutic agent is neither degraded nor excreted, but can be delivered to tissues, thus increasing its therapeutic effectiveness.
It has been proposed that compounds, such as peptides, peptide mimetics, and oligonucleotides, or analogs or derivatives thereof, can be used as potential therapeutic agents. However, problems have been encountered in administering such compounds to a subject. For example, proteases and endonucleases present throughout the body digest such compounds, severely decreasing their biological activity. Other problems involve the elicitation of an immune response against the compound resulting in the degradation and inactivation of such compounds, and rapid renal clearance, particularly if the therapeutic agent has a low molecular weight. Hence, in order to be effective, such therapeutic agents must be administered frequently, and parenterally rather than orally. An example of such a therapeutic agent is insulin, which is typically injected several times daily by diabetics.
In efforts to overcome these problems, researchers have attempted to modify chemically such therapeutic agents in order to manipulate their pharmacologic properties1, and perhaps enable them to survive longer in vivo before being degraded and removed from the blood stream. For example, one method of chemically modifying therapeutics is to append water-soluble polymer chains, such as polyethylene glycol (PEG), to the therapeutic agent2. Researchers have designed a PEG-lysine copolymer having multiple attachment sites3, and have conjugated the copolymer to low molecular weight therapeutic agents. However, such modifications have inherent limitations. For example, they frequently interfere with the bioavailability of the therapeutic. Consequently, if the target for a therapeutic agent is intracellular, and the modification of the therapeutic prevents its crossing of the cell membrane, then the bioavailability of the therapeutic agent is reduced due to the chemical modification.
Another limitation to attaching a water-soluble polymer to a therapeutic agent involves modulating the biological activity of the therapeutic agent in a deleterious manner. For example, if the modification of the therapeutic agent alters its three dimensional structure, then its ability to bind a receptor site it was designed to bind can be decreased, resulting in a decrease of activity.
Hence, what is needed is a carrier of a therapeutic agent that reduces the chance of an elicitation of an immune response against the therapeutic agent.
Moreover, what is needed is a carrier that protects therapeutic agents from protease/peptidase/nuclease degradation in vivo, thereby eliminating the need for repetitive administration of the therapeutic agent.
In addition, what is needed is a carrier of a therapeutic agent that enhances cellular trans-membrane delivery of the therapeutic agent.
Also, what is needed is a carrier of a therapeutic agent that does not release the therapeutic agent until the carrier has crossed the cell membrane, and once inside the cell, the carrier can release the therapeutic agent in a biologically active state.
What is also needed is a carrier of a therapeutic agent that does not interfere with the bioavailability of the therapeutic agent.
There is provided, in accordance with the present invention, a carrier for in vivo delivery of therapeutic agents that does not possess the shortcomings of other drug delivery carriers as described above, and offers the advantages of not interfering with the bioavailability of a therapeutic agent, protecting the therapeutic agent from proteolytic/nucleolytic degradation, from eliciting an immune response, and from rapid renal clearance, to name only a few.
Broadly, the present invention provides a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer, and at least one thiol compound conjugated to the polymer, such that the thiol group of the thiol compound and the thiol group of the therapeutic agent form a disulfide bond. The disulfide bond can be broken in a physiologically relevant reducing environment, such as that found in the cytosol of a cell. Hence, Applicants have discovered a way to modify a therapeutic agent to minimize degradation in vivo, and yet not limit its bioavailability. In addition, the present invention is particularly well suited to deliver therapeutic agents to the cytosol of cells. Glutathione, a naturally occurring reducing agent is found predominantly in cell cytosol. Hence, glutathione can reduce a disulfide bond, and release a therapeutic agent from a carrier of the present invention within the cells of the target tissue. In addition, since more than one thiol compound can be conjugated to the polymer, the carrier of the present invention can deliver more than one molecule of therapeutic agent to a target cell or tissue.
Numerous therapeutic agents comprise thiol groups, and can be used to conjugate the therapeutic agent to a carrier of the present invention with a disulfide bond. For example, therapeutic agents which are peptides comprising a cysteine residue can be delivered in vivo with a carrier of the present invention. In addition, analogs or derivatives of peptides which serve as therapeutic agents can be made to comprise a thiol group so that they can be delivered in vivo with a carrier of the present invention. Even nucleotides and analogs or derivatives thereof, used in antisense therapy for example, can be easily modified to comprise a thiol group in order to be carried via a carrier of the present invention.
Another example of a therapeutic agent comprising a thiol group, which can be conjugated to a carrier of the present invention for in vivo delivery, is a therapeutic agent which inhibits HIV-1 replication. More specifically, it has been determined that the HIV Tat protein strongly activates HIV transcription through its interactions with the TAR RNA region. The TAR RNA domain consists of the first 57 nucleotides of all virally encoded RNAs. The predicted TAR RNA secondary structure is a double-stranded stem with a 3-base bulge and a 6-base loop. HIV-1 Tat is a small nuclear protein containing 86-102 amino acids, and is encoded by multiply spliced mRNA. The 3-base bulge in TAR RNA and several other flanking nucleotides are essential for Tat-TAR interaction.
Tat protein apparently acts to promote transcription by binding through its basic domain to the 3-base bulge of TAR. This is accompanied by recruitment of host cellular factors, including Tat and TAR binding proteins, to the TAR RNA stem and 6-base loop, as well as to the complex of template DNA, transcription factors and RNA polymerase. Initiation of proviral gene expression appears to occur by activation of an NF-xcexaB and/or Sp1-dependent promoter, resulting in production of viral transcripts at a sufficient level to provide synthesis of Tat protein, which then interacts with TAR to allow enhanced production of elongated HIV transcripts.
Efforts have been made to develop a therapeutic agent which binds TAR, and blocks Tat-TAR binding. For example, a 10-residue Tat peptide with an appended 4-mer antisense oligonucleotide can specifically bind to TAR RNA, as shown by its ability to stimulate RNase H-mediated cleavage at the site of oligonucleotide annealing to the 6-base single-stranded loop. In another example, a biotinylated peptide has also been shown to inhibit Tat binding to TAR (please see Choudhury, I., Wang, J., Rabson, A. B., Stein, S., Pooyan, S., Stein, S. and Leibowitz, M. J. (1998) Inhibition of HIV-1 replication by a Tat RNA binding domain peptide analog. J. Acqu. Immune Def. Syndr. and Human Retrovirol., 17, 104-111, incorporated by reference herein in its entirety).
Hence, the present invention extends to a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer, and at least one thiol compound conjugated to the polymer, such that the thiol group of the thiol compound and the thiol group of the therapeutic agent form a disulfide bond, and the therapeutic agent is a Tat inhibitory polypeptide derivative. More particularly, in an embodiment of the invention, a therapeutic agent comprising a thiol group, which is a Tat-inhibitory binding peptide derivative, relates to biotinylated peptides of the formula I:
R-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-X-(biotin)-Cys-NH2xe2x80x83xe2x80x83(SEQ ID NO:1)
wherein R is the residue of the carboxylic acid, or an acetyl group, and X is a Cys or Lys residue, and analogs thereof, and biologically and pharmaceutically acceptable salts thereof, all stereo, optical and geometrical isomers thereof where such isomers exist, as well as the pharmaceutically acceptable salts and solvates thereof, which exhibit advantageous properties, including binding to TAR, inhibition of LTR-dependent reporter gene expression in a model cell assay and, finally, inhibition of HIV-1 replication, as determined as assays of HIV-induced syncytium formation, cytotoxicity and reverse transcriptase production. The biotinylated peptide of formula I can be readily conjugated to a carrier of the present invention via a disulfide bond between the sulfur of the thiol group of a Cys residue and the sulfur of the thiol group of the thiol compound of the carrier.
Examples of such Tat peptide derivatives include, but are not limited to:
In another embodiment, the present invention extends to a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer, and at least one thiol compound conjugated to the polymer, such that the thiol group of the thiol compound and the thiol group of the therapeutic agent form a disulfide bond, and the therapeutic agent is a Tat inhibitory polypeptide derivative comprising an amino acid sequence of:
N-acetyl-DCys-DLys-(biotin)-DArg-DArg-DArg-DGln-DArg-DArg-DLys-DLys-DArg-NH2xe2x80x83xe2x80x83(SEQ ID NO:8)
or analogs or derivatives thereof, and biologically and pharmaceutically acceptable salts thereof, which exhibit advantageous properties, including binding to xcex94TAR, inhibition of LTR-dependent reporter gene expression in a model cell assay and, finally, inhibition of HIV-1 replication.
Furthermore, the present invention extends to a method of treating a viral infection in a mammal in need of such treatment. More particularly, such a method comprises administering to a mammal a therapeutically effective amount of a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer, and at least one thiol compound conjugated to the polymer, such that the thiol group of the thiol compound and the thiol group of the therapeutic agent form a disulfide bond, and the therapeutic agent comprises a Tat-inhibitory binding peptide derivative which comprises a biotinylated peptide of the formula I:
R-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-X-(biotin)-Cys-NH2xe2x80x83xe2x80x83(SEQ ID NO:1)
wherein R is the residue of a carboxylic acid or acetyl group, X is a Cys or Lys residue, analogs thereof, and biologically and pharmaceutically acceptable salts thereof. Throughout the specification and appended claims, the polypeptide of formula I, and its analogs and salts, encompass all stereo, optical and geometrical isomers thereof where such isomers exist, as well as the pharmaceutically acceptable salts and solvates thereof. Where appropriate, the polypeptide or its analogs can be utilized as its corresponding amide form. The amino acid residues described herein are preferred to be in the xe2x80x9cLxe2x80x9d isomeric form. However, residues in the xe2x80x9cDxe2x80x9d isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
More particularly, a therapeutic agent having applications in such a method comprises an amino acid sequence including, but not limited to:
In another embodiment, the present invention extends to a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer, and at least one thiol compound conjugated to the polymer, such that the thiol group of the thiol compound and the thiol group of the therapeutic agent form a disulfide bond, and the therapeutic agent is a Tat inhibitory polypeptide derivative comprising an amino acid sequence as set forth below:
N-acetyl-DCys-DLys-(biotin)-DArg-DArg-DArg-DGln-DArg-DArg-DLys-DLys-DArg-NH2xe2x80x83xe2x80x83(SEQ ID NO:8)
or analogs or derivatives thereof, and biologically and pharmaceutically acceptable salts thereof, which exhibit advantageous properties, including binding to xcex94TAR, inhibition of LTR-dependent reporter gene expression in a model cell assay and, finally, inhibition of HIV-1 replication.
Moreover, the present invention extends to a method of utilizing a carrier of the present invention conjugated to a peptide comprising an amino acid sequence as set forth in SEQ ID NO:8, and biologically and pharmaceutically acceptable salts thereof, to treat a viral infection in a mammal in need of such treatment. Such a method comprises administering to a mammal a therapeutically effective amount of a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer, and at least one thiol compound conjugated to the polymer, such that the thiol group of the thiol compound and the thiol group of the therapeutic agent form a disulfide bond, and the therapeutic agent comprises a biotinylated peptide comprising an amino acid sequence as set forth in SEQ ID NO:8.
In particular, methods of the present invention stated above can be used to treat retroviral infections such as AIDS, in humans.
Moreover, the invention extends to a carrier for in vivo delivery of a therapeutic agent, comprising a polymer which has a branched or linear structure. For purposes of this application, the term xe2x80x9cpolymerxe2x80x9d encompasses both homopolymers and copolymers. Preferably, the polymer is a water soluble polymer.
Examples of water soluble polymers which have applications in the present invention include, but are not limited to, polyethylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, polyaminoacids (homopolymers), polypropylene glycol, copolymers of ethylene glycol/propylene glycol, ethylene/maleic anhydride copolymer, polyaminoacids, copolymer of polyethylene glycol and an amino acid, polypropylene oxide/ethylene oxide copolymers, or polyethylene glycol/thiomalic acid copolymers. In a preferred embodiment, the polymer comprises a copolymer of polyethylene glycol and lysine.
Moreover, a thiol compound can be conjugated to the polymer of the carrier, or conjugated to at least one functional group attached to the polymer which is available for reaction with the thiol compound, provided the thiol group of the thiol compound is available to form a disulfide bond with a thiol group of the therapeutic agent.
Numerous functional groups can be attached to a polymer of a carrier of the present invention and used to conjugate a thiol compound thereto. Moreover, more than one type of functional group can be concurrently attached to the polymer, and available to conjugate a thiol compound to the polymer. Examples of functional groups having applications in this embodiment of the present invention include, but are not limited to, ketones, esters, carboxylic acids, aldehydes, alcohols, thiols, or amines. In a preferred embodiment, the polyethylene glycol/lysine copolymer has a carboxylic acid group attached thereto, and available for reaction. Hence, more than one molecule of therapeutic agent can be conjugated to a carrier of the present invention, and delivered to a target cell or tissue.
A polymer of a carrier of the present invention can have any molecular weight. In an embodiment of the invention, the polymer has a molecular weight range of about 1,000 to about 1,000,000 Daltons, and preferably a molecular weight range of about 20,000 to 200,000 Daltons. In a preferred embodiment, the polymer has a molecular weight of about 27,000 Daltons.
The present invention further extends to a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer, and at least one thiol compound conjugated to the polymer at an interval so that the thiol group of the at least one thiol compound and a thiol group of a therapeutic agent can form a disulfide bond. As used herein, the term xe2x80x9cintervalxe2x80x9d indicates a distance between thiol compounds conjugated to the polymer of a carrier of the present invention. In an embodiment, the interval between thiol compounds conjugated to the polymer is about 100 to about 10,000 Daltons. In a preferred embodiment, the interval between conjugation of thiol compounds to a polymer of the carrier is about 300 to about 3,000 Daltons.
Furthermore, the present invention extends to a carrier comprising a polymer with functional groups attached thereto, wherein the functional groups are available for conjugation to a thiol compound, and are attached to the polymer at an interval. In an embodiment, the interval between functional groups attached to the polymer is about 100 to about 10,000 Daltons. In a preferred embodiment, the interval between functional groups attached to a polymer of the carrier is about 300 to about 3,000 Daltons.
Moreover, examples of thiol compounds having applications in a carrier of the present invention include, but are not limited to, cysteamine, 1-amino-2-methyl-2-propanethiol, or 1-amino-2-propanethiol, to name only a few.
In addition, the present invention extends to a carrier for in vivo delivery of a therapeutic agent having a thiol group, wherein the carrier further comprises a cell uptake promoter conjugated to the polymer. The cell uptake promoter enhances the ability of the carrier with the therapeutic agent conjugated thereto, to cross a cell membrane and enter the cell""s cytosol.
Numerous cell uptake promoters are known, and have applications in embodiments of the present invention. An example of a cell uptake promoter having applications therein is biotin. Hence, conjugation of biotin to a polymer of a carrier of the present invention enhances the ability of the carrier, and the therapeutic agent attached thereto via a disulfide bond, to cross a cell membrane and enter a cell""s cytosol. Once inside the cell, the disulfide bond between the therapeutic agent and the carrier is reduced, and the therapeutic agent is released to act upon its target.
Moreover, it has been discovered that the cell uptake promoter need not be conjugated only to the polymer in order to be effective. Rather, the cell uptake promoter can also be conjugated to the therapeutic agent, and still enhance the ability of the carrier and the therapeutic agent to cross a cell membrane.
The present invention further extends to methods of making a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer and at least one thiol compound conjugated to the polymer, such that the thiol group of the thiol compound and the thiol group of the therapeutic agent form a disulfide bond. One such method disclosed herein comprises the steps of:
a) reacting a thiol compound with a disulfide compound to form a first intermediate wherein the thiol group of the thiol compound and a sulfur atom of the disulfide bond of the disulfide compound form a disulfide bond; and
b) reacting the first intermediate with a polymer to form the carrier, wherein the first intermediate is conjugated to the polymer, so that the sulfur atom of the thiol compound of the carrier and the thiol group of the therapeutic agent can form a disulfide bond.
Any disulfide compound can be used in step (a) of the method recited above. Preferably, the disulfide compound is symmetric. In an embodiment of the present invention, the disulfide compound is 2,2xe2x80x2-dithiodipyridine.
Another method of making a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, as set forth herein, comprises the steps of:
a) reacting a first thiol compound with a second thiol compound to form a first intermediate comprising a disulfide bond;
b) reacting the first intermediate with a polymer to form a second intermediate, wherein the first intermediate is conjugated to the polymer;
c) reducing the disulfide bond to form a third intermediate comprising the polymer and the first thiol compound conjugated to the polymer so that the thiol group of the first thiol compound is available for reaction; and
d) reacting the third intermediate with a disulfide compound to form the carrier, wherein the thiol group of the first thiol compound and a sulfur atom of the disulfide bond of the disulfide compound form a disulfide bond.
Under appropriate conditions, the sulfur atom of the first thiol compound and the thiol group of the therapeutic agent can form a disulfide bond.
In an embodiment of the present invention, the first thiol compound and the second thiol compound are the same compound.
Numerous thiol compounds have applications in methods of the present invention. Examples include cysteamine, 1-amino-2-methyl-2-propanethiol, and 1-amino-2-propanethiol, to name only a few.
Moreover, any disulfide compound can be used in step (d) of this method. Preferably, the disulfide compound is symmetric. In an embodiment of the present invention, the disulfide compound is 2,2xe2x80x2-dithiodipyridine.
In an embodiment of the present invention, a therapeutic agent comprising a thiol group is a Tat-inhibitory peptide derivative, and comprises a biotinylated peptide of the formula I:
R-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-X-(biotin)-Cys-NH2xe2x80x83xe2x80x83(SEQ ID NO:1)
wherein R is the residue of a carboxylic acid or an acetyl group, and X is a Cys or Lys residue, and analogs thereof, and the biologically and pharmaceutically acceptable salts thereof, and all stereo, optical and geometrical isomers thereof where such isomers exist, as well as the pharmaceutically acceptable salts and solvates thereof. Examples of analogs of such a therapeutic agent include, but are not limited to:
In another embodiment of the present invention, a therapeutic agent comprising a thiol group is a Tat-inhibitory peptide derivative comprising an amino acid sequence of SEQ ID NO:8:
N-acetyl-DCys-DLys-(biotin)-DArg-DArg-DArg-DGln-DArg-DArg-DLys-DLys-DArg-NH2xe2x80x83xe2x80x83(SEQ ID NO:8)
analogs or derivatives thereof, as well as all pharmaceutically acceptable salts thereof.
The production of such agents can readily be accomplished with presently known method of producing peptide including, but not limited to solid phase synthesis of peptides. Moreover, it is also readily apparent to one skilled in the art on methods of biotinylating such peptides.
The present invention further extends to methods of making a carrier for in vivo delivery of a therapeutic agent, as set forth above, wherein the polymer has a branched or linear structure. Preferably, the polymer is a water soluble polymer. Examples of water soluble polymers having applications herein include polyethylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, polyaminoacids (homopolymers), polypropylene glycol, copolymers of ethylene glycol/propylene glycol, ethylene/maleic anhydride copolymer, polyaminoacids, copolymer of polyethylene glycol and an amino acid, polypropylene oxide/ethylene oxide copolymers, or polyethylene glycol/thiomalic acid copolymers.
Moreover, a polymer used in methods of the present invention can have any molecular weight. In an embodiment of the present invention, a polymer has a molecular weight range of about 1,000 to about 1,000,000 Daltons, and preferably a molecular weight range of about 20,000 to 200,000 Daltons. In a preferred embodiment, the polymer has a molecular weight of about 27,000 Daltons.
The present invention also extends to methods of making a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, comprising a polymer and at least one thiol compound conjugated to the polymer at an interval. In an embodiment of the invention, the interval between thiol compounds conjugated to the polymer is about 100 to about 10,000 Daltons, and preferably about 300 to about 3,000 Daltons.
The present invention further extends to methods for making carriers for in vivo delivery of a therapeutic agent comprising a thiol group, further comprising the step of conjugating a cell uptake promoter to the polymer prior to reacting the polymer with an intermediate. The cell uptake promoter enhances the ability of the carrier bound to the therapeutic agent to cross a cell membrane and enter the cell""s cytosol.
Numerous cell uptake promoters are known, and have applications in the present invention. An example of a cell uptake promoter is biotin. Hence, conjugating biotin to a polymer of a carrier of the present invention enhances the ability of the carrier and the therapeutic agent conjugated thereto, to cross a cell membrane and enter a cell""s cytosol. As explained above, once the carrier crosses the cell membrane and enters the cytosol, the disulfide bond is reduced, and agent is released to act on its target.
Moreover, it has been discovered that the cell uptake promoter need not be attached only to the polymer. Rather, the cell uptake promoter can also be conjugated to the therapeutic agent, and still enhance the ability of the carrier and the therapeutic agent to cross a cell membrane.
The present invention further extends to a method of making a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer comprising at least one functional group attached thereto, and a thiol compound conjugated to the at least one functional group, such that the thiol group of the thiol compound and the thiol group of the therapeutic agent form a disulfide bond. An example of a method for making such a carrier comprises the steps of:
a) reacting a thiol compound with a disulfide compound to form a first intermediate wherein the thiol group of the thiol compound and a sulfur atom of the disulfide bond of the disulfide compound form a disulfide bond; and
b) reacting the first intermediate with a polymer comprising at least one functional group attached thereto to form the carrier, wherein the first intermediate is conjugated to the functional group so that the sulfur atom of the thiol compound and the thiol group of the therapeutic agent can form a disulfide bond under appropriate conditions.
Furthermore, the present invention extends to a method of making a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, as set forth herein, comprising the steps of:
a) reacting a first thiol compound with a second thiol compound to form a first intermediate, wherein the thiol group of the first thiol compound and the thiol group of the second thiol compound form a disulfide bond;
b) reacting the first intermediate with a polymer comprising at least one functional group attached thereto to form a second intermediate, wherein the first intermediate is conjugated to the at least one functional group;
c) reducing the disulfide bond of the second intermediate to form a third intermediate comprising the polymer with at least one functional group and the first thiol compound conjugated to the functional group, so that the thiol group of the first thiol compound able for reaction;
d) reacting the third intermediate with a disulfide compound to form the carrier, wherein the thiol group of the first thiol compound and a sulfur atom of the disulfide bond of the disulfide compound form a disulfide bond. Hence, under appropriate conditions, the disulfide bond of the carrier can be reduced, and the sulfur atom of the first thiol compound and the thiol group of the therapeutic agent can form a disulfide bond.
In an embodiment of the present invention, the first thiol compound and the second thiol compound can be the same compound.
The present invention extends to methods of making a carrier of the present invention, as set forth above, wherein the at least one functional group attached to a polymer used in methods of the present invention comprises a ketone, an ester, a carboxylic acid, an aldehyde, an alcohol, a thiol, or an amine, to name only a few.
Moreover, the present invention extends to methods of making a carrier of the present invention as described above, wherein the at least one functional group is attached to the polymer of a carrier of the present invention at an interval. In an embodiment of the invention, the interval between functional groups attached to the polymer is about 100 to about 10,000 Daltons, and preferably about 300 to about 3,000 Daltons.
As stated above, a therapeutic agent conjugated to a carrier of the present invention comprises at least one thiol group. In an example, a therapeutic agent conjugated to a carrier of the present invention comprises a Tat inhibitory peptide derivative of the formula I:
R-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-X-(biotin)-Cys-NH2xe2x80x83xe2x80x83(SEQ ID NO:1)
wherein R is the residue of a carboxylic acid or an acetyl group, and X is a Cys or Lys residue, and analogs thereof, and the biologically and pharmaceutically acceptable salts thereof, along with all stereo, optical and geometrical isomers thereof, where such isomers exist, and pharmaceutically acceptable salts and solvates thereof.
Moreover, examples of analogs of such a therapeutic agent having applications in the present invention include, but are not limited to:
to name only a few.
In another embodiment, a therapeutic agent comprising a thiol group and conjugated to a carrier of the present invention, comprises an amino acid sequence as set forth in SEQ ID NO:8:
N-acetyl-DCys-DLys-(biotin)-DArg-DArg-DArg-DGln-DArg-DArg-DLys-DLys-DArg-NH2xe2x80x83xe2x80x83(SEQ ID NO:8).
or analogs or derivatives thereof, and pharmaceutically acceptable salts thereof.
Applicants have also discovered that the release rate of a therapeutic agent from a carrier of the present invention can be modulated, depending on the steric hindrance of the thiol compound of the carrier. In particular, Applicants have discovered that the greater the steric hindrance of the thiol compound, the slower the release rate of the therapeutic agent from the carrier. Hence, the present invention extends to a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer and at least one thiol compound comprising at least one functional group, conjugated to the polymer, such that the thiol group of the thiol compound and the thiol group of the therapeutic agent can form a disulfide bond, and the rate at which the disulfide bond is reduced in an appropriate physiological environment is dependent on the size of the at least one functional group attached to the thiol compound.
Moreover, the present invention extends to methods of making a carrier of the present invention as described above, wherein the at least one functional group is attached to the polymer of a carrier of the present invention at an interval. In an embodiment of the invention, the interval between functional groups attached to the polymer is about 100 to about 10,000 Daltons, and preferably about 300 to about 3,000 Daltons.
The present invention further extends to another embodiment wherein the release rate of a therapeutic agent from a carrier of the present invention can be modulated depending on the steric hindrance of the thiol compound of the carrier. In particular, disclosed herein is a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer and at least one thiol compound comprising at least one functional group, and a polymer comprising at least one functional group available for reaction, so that the thiol compound is conjugated to the at least one functional group attached to the polymer, and the thiol group of the thiol compound and the thiol group of the therapeutic agent can form a disulfide bond. The rate of reduction of the disulfide bond linking the therapeutic agent to the carrier is dependent on the size of the at least one functional group attached to the thiol compound.
Accordingly, it is a principal object of the present invention to provide a carrier for in vivo delivery of a therapeutic agent which can be conjugated to a carrier via a disulfide bond.
It is a further object of the invention to provide a carrier for in vivo delivery of a therapeutic agent, wherein the therapeutic agent is conjugated to the carrier via a biodegradable disulfide bond which is difficult to reduce in extracellular fluids in vivo.
It is a further object of the present invention to provide a carrier for in vivo delivery of a therapeutic agent which will remain substantially conjugated to the carrier until the carrier crosses a cell membrane. Once inside the cell, the cytosolic environment reduces the disulfide bond conjugating the therapeutic agent to the carrier, and the therapeutic agent is released to act on its target.
It is a further object of the present invention to provide a carrier for in vivo delivery of a therapeutic agent which increases the bioavailability of the therapeutic agent so that the rate of repeated parenteral administration of the therapeutic agent can be minimized.
It is yet another object of the present invention to provide methods of modulating the release of a therapeutic agent in vivo wherein the release rate of the therapeutic agent from the carrier is dependent upon three dimensional configuration of a thiol compound used in methods of making carriers of the present invention.
Yet still another object of the present invention is to provide a carrier for in vivo delivery of a therapeutic agent comprising a thiol group which has a structure that can reduce the rate at which the therapeutic agent is released from the carrier. This object can be used to cause the timed release of the therapeutic agent in vivo.
It is yet another object of the present invention to provide a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the therapeutic agent is a Tat-inhibitory polypeptide derivative, analogs thereof, or biologically and pharmaceutically acceptable salts thereof.
It is yet another object of the present invention to provide a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the therapeutic agent is a Tat-inhibitory peptide derivative, and administration of a carrier of the present invention conjugated to a Tat-inhibitory peptide derivative disclosed herein can be used to treat a mammal suffering from a retroviral infection, such as AIDS.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.